Potential impact of efflux pump genes in mediating rifampicin resistance in clinical isolates of Mycobacterium tuberculosis from India

Autoři: Anshika Narang aff001;  Kushal Garima aff001;  Shraddha Porwal aff001;  Archana Bhandekar aff001;  Kamal Shrivastava aff001;  Astha Giri aff001;  Naresh Kumar Sharma aff001;  Mridula Bose aff001;  Mandira Varma-Basil aff001
Působiště autorů: Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India aff001
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223163


Despite the consideration of chromosomal mutations as the major cause of rifampicin (RIF) resistance in M. tuberculosis, the role of other mechanisms such as efflux pumps cannot be ruled out. We evaluated the role of four efflux pumps viz., MmpL2 (Rv0507), MmpL5 (Rv0676c), Rv0194 and Rv1250 in providing RIF resistance in M. tuberculosis. The real time expression of the efflux pumps was analyzed in 16 RIF resistant and 11 RIF susceptible clinical isolates of M. tuberculosis after exposure to RIF. Expression of efflux pumps in these isolates was also correlated with mutations in the rpoB gene and MICs of RIF in the presence and absence of efflux pump inhibitors. Under RIF stress, Rv0194 was induced in 8/16 (50%) RIF resistant and 2/11 (18%) RIF susceptible isolates; mmpL5 in 7/16 (44%) RIF resistant and 1/11 (9%) RIF susceptible isolates; Rv1250 in 4/16 (25%) RIF resistant and 2/11 (18%) RIF susceptible isolates; and mmpL2 was upregulated in 2/16 (12.5%) RIF resistant and 1/11 (9%) RIF susceptible isolates. This preliminary study did not find any association between Rv0194, MmpL2, MmpL5 and Rv1250 and RIF resistance. However, the overexpression of Rv0194 and mmpL5 in greater number of RIF resistant isolates as compared to RIF susceptible isolates and expression of Rv0194 in wild type (WT) resistant isolates suggests a need for further investigations.

Klíčová slova:

Extensively drug-resistant tuberculosis – Gene expression – Hyperexpression techniques – India – Mutation – Tuberculosis – RNA isolation


1. Mitchison DA, Nunn AJ. Influence of initial drug resistance on the response to short-course chemotherapy of pulmonary tuberculosis. Am. Rev. Respir. Dis. 1986; 133:423–430. doi: 10.1164/arrd.1986.133.3.423 2420242

2. Somoskovi A, Parsons LM, Salfinger M. The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respir Res. 2001;2(3):164–168. doi: 10.1186/rr54 11686881

3. Felmlee TA, Liu Q, Whelen AC, Williams D, Sommer SS, Persing DH. Genotypic detection of Mycobacterium tuberculosis rifampicin resistance: Comparison of single-strand conformation polymorphism and dideoxy fingerprinting. J. Clin Microbiol. 1995; 33:1617–1623. 7650198

4. Garcia de Viedma D, del Sol Diaz Infantes M, Lasala F, Chaves F, Alcala L, Bouza E. New realtime PCR able to detect in a single tube multiple rifampin resistance mutations and high-level isoniazid resistance mutations in Mycobacterium tuberculosis. J Clin Microbiol. 2002;40(3):988–995. doi: 10.1128/JCM.40.3.988-995.2002 11880428

5. Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A, et al. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell. 2001;104:901–912. doi: 10.1016/s0092-8674(01)00286-0 11290327

6. Betts JC, Lukey PT, Robb LC, McAdam RA, Duncan K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol. 2002;43:717–731. doi: 10.1046/j.1365-2958.2002.02779.x 11929527

7. Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis. 1998;79(1):3–29. doi: 10.1054/tuld.1998.0002 10645439

8. Heym B, Cole ST. Multidrug resistance in Mycobacterium tuberculosis. Int J Antimicrob Agents. 1997;8(1):61–70. 18611785

9. Banerjee SK, Bhatt K, Misra P, Chakraborti PK. Involvement of a natural transport system in the process of efflux-mediated drug resistance in M. smegmatis. Mol Gen Genet. 2000;262(6):949–956. doi: 10.1007/pl00008663 10660056

10. Siddiqi N, Das R, Pathak N, Banerjee S, Ahmed N, Katoch VM et al. Mycobacterium tuberculosis isolate with a distinct genomic identity overexpresses a tap-like efflux pump. Infection. 2004;32(2):109–111. doi: 10.1007/s15010-004-3097-x 15057575

11. Gupta AK, Chauhan DS, Srivastava K, Das R, Batra S, Mittal M et al. Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria. J Commun Dis. 2006;38(3):246–254. 17373356

12. Jiang X, Zhang W, Zhang Y, Gao F, Lu C, Zhang X et al. Assessment of efflux pump gene expression in a clinical isolate Mycobacterium tuberculosis by real-time reverse transcription PCR. Microb Drug Resist. 2008;14:7–11. doi: 10.1089/mdr.2008.0772 18321205

13. Pang Y, Lu J, Wang Y, Song Y, Wang S, Zhao Y. Study of the rifampin monoresistance mechanism in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2013;57(2):893–900. doi: 10.1128/AAC.01024-12 23208715

14. Kent PT, Kubica GP. Public health mycobacteriology: A guide for the level III laboratory. Centers for Diseases Control, Atlanta. 1985.

15. Varma-Basil M, Garima K, Pathak R, Dwivedi SK, Narang A, Bhatnagar A et al. Development of a novel PCR restriction analysis of the hsp65 gene as a rapid method to screen for the Mycobacterium tuberculosis complex and nontuberculous mycobacteria in high-burden countries. J ClinMicrobiol. 2013;51(4):1165–1170.

16. Standard operating procedures for C&DST labs. Central TB Division [Internet]. [cited 10 January 2016]. Available from: https://tbcindia.gov.in

17. Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A et al. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol. 1998;36(2):362–366. 9466742

18. Huitric E, Werngren J, Juréen P, Hoffner S. Resistance levels and rpoB gene mutations among in vitro-selected rifampin-resistant Mycobacterium tuberculosis mutants. Antimicrob Agents Chemother. 2006;50(8):2860–2862. doi: 10.1128/AAC.00303-06 16870787

19. Gupta AK, Katoch VM, Chauhan DS, Sharma R, Singh M, Venkatesan K et al. Microarray analysis of efflux pump genes in multidrug-resistant Mycobacterium tuberculosis during stress induced by common anti-tuberculous drugs. Microb Drug Resist. 2010;16(1):21–28. doi: 10.1089/mdr.2009.0054 20001742

20. Garima K, Pathak R, Tandon R, Rathor N, Sinha R, Bose M et al. Differential expression of efflux pump genes of Mycobacterium tuberculosis in response to varied subinhibitory concentrations of antituberculosis agents. Tuberculosis (Edinb). 2015;95(2):155–161.

21. Louw GE, Warren RM, Gey van Pittius NC, Leon R, Jimenez A, Hernandez-Pando R, McEvoy CR et al. Rifampicin reduces susceptibility to ofloxacin in rifampicin-resistant Mycobacterium tuberculosis through efflux. Am J Respir Crit Care Med. 2011;184(2):269–276. doi: 10.1164/rccm.201011-1924OC 21512166

22. Osdd.net. Open Source Drug Discovery [Internet]. [cited 9 April 2013]. Available from: http://www.osdd.net.

23. Genolist.pasteur.fr. TubercuList Web Server [Internet]. 2008 [cited 9 March 2013]. Available from: http://genolist.pasteur.fr/TubercuList/.

24. Lam TH, Yuen KY, Ho PL, Wong KC, Leong WM, Law HK et al. Differential fadE28 expression associated with phenotypic virulence of Mycobacterium tuberculosis. Microb Pathog. 2008;45(1):12–17. doi: 10.1016/j.micpath.2008.01.006 18486437

25. Masiewicz P, Brzostek A, Wolański M, Dziadek J, Zakrzewska-Czerwińska J. A novel role of the PrpR as a transcription factor involved in the regulation of methylcitrate pathway in Mycobacterium tuberculosis. PLoS One. 2012;7(8):e43651. doi: 10.1371/journal.pone.0043651 22916289

26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–408. doi: 10.1006/meth.2001.1262 11846609

27. Heymann SJ, Brewer TF, Wilson ME, Fineberg HV. The need for global action against multidrug-resistant tuberculosis. JAMA 1999; 281:2138–2140. doi: 10.1001/jama.281.22.2138 10367825

28. Gillespie SH, Billington OJ, Breathnach A, McHugh TD. Multiple drug-resistant Mycobacterium tuberculosis: evidence for changing fitness following passage through human hosts. Microb Drug Resist. 2002;8(4):273–279. doi: 10.1089/10766290260469534 12523624

29. Ohno H, Koga H, Kohno S, Tashiro T, Hara K. Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrob Agents Chemother. 1996;40(4):1053–1056. 8849230

30. Feuerriegel S, Oberhauser B, George AG, Dafae F, Richter E, Rüsch-Gerdes S et al. Sequence analysis for detection of first-line drug resistance in Mycobacterium tuberculosis strains from a high-incidence setting BMC Microbiol. 2012;12: 90. doi: 10.1186/1471-2180-12-90 22646308

31. Wilson M, De Risi J, Kristensen HH, Imboden P, Rane S, Brown PO et al. Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization. ProcNatlAcadSci U S A. 1999;96(22):12833–12838.

32. Betts JC, McLaren A, Lennon MG, Kelly FM, Lukey PT, Blakemore SJ et al. Signature gene expression profiles discriminate between isoniazid-, thiolactomycin-, and triclosan-treated Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2003;47(9):2903–2913. doi: 10.1128/AAC.47.9.2903-2913.2003 12936993

33. Fu LM, Tai SC. The Differential Gene Expression Pattern of Mycobacterium tuberculosis in Response to Capreomycin and PA-824 versus First-Line TB Drugs Reveals Stress- and PE/PPE-Related Drug Targets. Int J Microbiol. 2009;2009:879621. doi: 10.1155/2009/879621 20016672

34. Li G, Zhang J, Guo Q, Jiang Y, Wei J, Zhao LL et al. Efflux pump gene expression in multidrug-resistant Mycobacterium tuberculosis clinical isolates. PLoS One. 2015;10(2):e0119013. doi: 10.1371/journal.pone.0119013 25695504

35. Calgin MK, Sahin F, Turegun B, Gerceker D, Atasever M, Koksal et al. Expression analysis of efflux pump genes among drug-susceptible and multidrug-resistant Mycobacterium tuberculosis clinical isolates and reference strains. Diagn Microbiol Infect Dis. 2013;76(3):291–7. doi: 10.1016/j.diagmicrobio.2013.02.033 23561272

36. Ahmad S, Mokaddas E, Fares E. Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai. Diagn Microbiol Infect Dis. 2002;44(3):245–252. doi: 10.1016/s0732-8893(02)00457-1 12493171

37. Deepa P, Therese KL, Madhavan HN. Detection and Characterization of mutations in rifampicin resistant Mycobacterium tuberculosis clinical isolates by DNA sequencing. Indian J Tuberc 2005;52:132–136.

38. McCammon MT, Gillette JS, Thomas DP, Ramaswamy SV, Graviss EA, Kreiswirth BN et al. Detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis using denaturing gradient gel electrophoresis. Antimicrob Agents Chemother. 2005;49(6):2200–2209. doi: 10.1128/AAC.49.6.2200-2209.2005 15917513

39. Ahmad S, Al-Mutairi NM, Mokaddas E. Variations in the occurrence of specific rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolates from patients of different ethnic groups in Kuwait. Indian J Med Res. 2012;135(5):756–762. 22771609

40. Qazi O, Rahman H, Tahir Z, Qasim M, Khan S, Ahmad Anjum A et al. Mutation pattern in rifampicin resistance determining region of rpoB gene in multidrug-resistant Mycobacterium tuberculosis isolates from Pakistan. Int J Mycobacteriol. 2014;3(3):173–177. doi: 10.1016/j.ijmyco.2014.06.004 26786485

41. Caleffi-Ferracioli KR, Amaral RC, Demitto FO, Maltempe FG, Canezin PH, Scodro RB et al. Morphological changes and differentially expressed efflux pump genes in Mycobacterium tuberculosis exposed to a rifampicin and verapamil combination. Tuberculosis (Edinb). 2016;97:65–72.

42. Pule CM, Sampson SL, Warren RM, Black PA, van Helden PD, Victor TC et al. Efflux pump inhibitors: targeting mycobacterial efflux systems to enhance TB therapy. J Antimicrob Chemother. 2016;71(1):17–26. doi: 10.1093/jac/dkv316 26472768

43. Gupta S, Tyagi S, Almeida DV, Maiga MC, Ammerman NC, Bishai WR. Acceleration of tuberculosis treatment by adjunctive therapy with verapamil as an efflux inhibitor. Am J Respir Crit Care Med. 2013;188(5):600–7. doi: 10.1164/rccm.201304-0650OC 23805786

44. Machado D, Couto I, Perdigão J, Rodrigues L, Portugal I, Baptista P et al. Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis. PLoS One. 2012;7(4):e34538. doi: 10.1371/journal.pone.0034538 22493700

45. Danilchanka O, Mailaender C, Niederweis M. Identification of a novel multidrug efflux pump of Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2008;52(7):2503–2511. doi: 10.1128/AAC.00298-08 18458127

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